The mosquito Anopheles gambiae is the primary vector of the deadly human malaria parasite Plasmodium falciparum in sub-Saharan Africa. While many laboratory studies have focused on how the mosquito immune system responds to and destroys these parasites, there is currently little available information on whether these responses are important under natural conditions. To address this need, the Luckhart laboratory is currently mapping single nucleotide polymorphisms (SNPs) in An. gambiae immune signaling genes to identify significant associations with P. falciparum infection in naturally occurring mosquito populations in endemic areas of Mali and Cameroon. In previous publications, the Luckhart laboratory demonstrated that human insulin in the blood meal and the insulin and insulin-like growth factor signaling (IIS) cascade can regulate malaria parasite development in Anopheles stephensi, a species closely related to An. gambiae. Hence, the IIS cascade proteins have been a major focus of these genotyping efforts. Thus far, we have identified numerous SNPs in the An. gambiae insulin-like peptide (AgILP) genes, including one in AgILP3 that is significantly associated with P. falciparum infection (Horton et al. 2010). Since this work, we have identified additional AgILP SNPs, including three SNPs in AgILP3 and AgILP4 that are predicted to alter protein function. Genotyping efforts are currently underway to determine whether any or all of these SNPs are also significantly associated with parasite infection in field collected mosquitoes. In light of data that demonstrate that human insulin and infection with P. falciparum can also induce the expression of ILP genes in the A. stephensi midgut, our genotyping data suggest that the AgILPs are involved in the regulation of P. falciparum infection under natural conditions. Based on these observations, we hypothesize that mosquito ILPs are produced in response to parasite infection, perhaps to amplify an earlier response to blood-derived factors or to insulin-like parasite factors, for sustained regulation of P. falciparum infection by the mosquito host. To test this hypothesis, we will determine which ILPs influence malaria parasite development in the mosquito midgut (Specific Aim 1), which pathways regulate ILP synthesis, secretion, and bioactivity (Specific Aim 2), and the effects of naturally occurring SNP mutations on ILP function (Specific Aim 3). These studies will not only provide key insights regarding immune cells signaling that is linked to parasite transmission under natural conditions, but will also provide novel targets for manipulating the mosquito immune response to reduce malaria transmission capacity. PUBLIC HEALTH RELEVANCE: Malaria is one of the leading causes of death from infectious diseases worldwide. The mosquitoes Anopheles stephensi and Anopheles gambiae are major vectors of the causative Plasmodium agents in India and Sub-Saharan Africa, respectively. Due to emerging challenges such as drug resistance in Plasmodium and insecticide resistance in the mosquito, there is an increasing need for novel malaria control strategies. The proposed project will identify and characterize new targets for genetic manipulation of the immune response in two major malaria vectors, with the potential of extending these findings to other Anopheles species in order to enhance malaria control.